Project Summary/Abstract Multicellular organisms require a high degree of coordination between cells and tissues in order to execute complex developmental processes and organ functions. This coordination is achieved by communication mediated by chemical signals such as hormones and growth factors, electrical signals such as action potentials, and mechanical signals. Mechanical signals can be generated within cells, for example by regulation of myosin-dependent contractility, or externally, for example by modulation of extracellular matrix composition. Organs of the respiratory, cardiovascular, and urogenital systems experience mechanical stress as part of their normal physiological landscape. For the cells within those organs, mechanical force influences many processes such as cell adhesion, cell motility, cell proliferation, cell survival, stem cell lineage commitment, and morphogenetic movements during development. A major current challenge in cell biology is to understand how cells sense and respond to mechanical cues to achieve diverse physiological responses. By studying cultured cells, it is possible to study the response of cells to mechanical stimulation delivered in a controlled fashion. Exposure of fibroblasts to uniaxial cyclic stretch results in a dramatic Stress Fiber- Remodeling, Repair, and Reinforcement (SF-R3) response. The SF-R3 response is essential for tensional homeostasis and protects the actin cytoskeleton from force-induced fragmentation. Force-induced changes in the actin cytoskeleton in turn provide information about the mechanical environment, promoting mechanosensitive changes in gene expression and other changes in cell behavior. Proteins containing double zinc fingers, called LIM domains, have recently been shown to play a central role in the response of cells to mechanical stress. The proposed research will employ a transdisciplinary approach incorporating genetic manipulations, biochemistry, cell behavioral assays, and quantitative fluorescence imaging to probe the mechanism by which physical forces result in changes of cell structure and function. The first aim will define the features of LIM domains that support their ability to recognize actin filaments that are exposed to mechanical stress. The second aim probes the mechanism by which a MAP-kinase cascade that is activated by mechanical stress influences effector proteins that are critical for the cellular response to mechanical cues. The third aim will explore the mechanism by which mechanical cues and the resulting changes in actin dynamics are communicated to the cell nucleus to affect changes in transcriptional output. The proposed research will expand our understanding of how mechanical force influences cell physiology and function.